Co-Infection Rates between SARS-CoV-2 and RSV in Oropharyngeal, Nasopharyngeal Aspirate and Saliva Samples of COVID-19 Patients, Shiraz, South of Iran

Statement of the Problem: Determining the prevalence of respiratory viruses' coinfection with coronavirus disease 2019 (COVID-19) is essential to defining its true clinical influence. Purpose: This study aimed to evaluate co-infection rates between severe acute respiratory syndrome–related coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV) in infected patients in Shiraz, south of Iran. Materials and Method: In a cross-sectional descriptive study, oropharyngeal, nasopharyngeal aspirate (NPA), and saliva samples of 50 COVID-19 patients who were referred to Ali-Asghar hospital (Shiraz, Iran) from March to August 2020, were collected. A control group consisted of age and sex-matched healthy participants. The nasopharyngeal and oropharyngeal aspirates were collected by sterile swabs. All cases were hospitalized, and all SARS-CoV-2 patients had a fever and respiratory symptoms. The samples were packed in a vial with 1 mL of transport medium and transported to the Valfagre specialty laboratory, where they were tested for RSV using a real-time polymerase chain reaction (PCR). Results: 100 nasopharyngeal/oropharyngeal aspirates and saliva samples including 50 healthy controls (24 females, 26 males) and 50 COVID-19 patients' samples (27 males and 23 females) were studied. There was no significant difference regarding age as well as gender between both groups (p> 0.05). None of the healthy subjects was infected with RSV; however, 5(10%) patients from COVID-19 group were infected with the RSV virus. Chi-square test did not show a significant difference between RSV infection in COVID-19 patients and healthy subjects. Conclusion: The outcome of present research showed that concurrent RSV with COVID 19 infection might be seen in hospitalized patients in Shiraz Southwest of Iran. For more reliable findings, further research on bigger populations, including more pathogens in several places around the country, and considering the severity of symptoms is required.


Introduction
Since December 2019, coronavirus disease 2019 (COV-ID- 19) virulent disease has spread worldwide. Asia has had the majority of the reported cases of COVID-19 followed by Europe, North America, and Australia.
Most of them were older than 18 years of age, and men consisted of 61.9% of the reported cases. Early patients occurred mostly amongst tourists from China and those who have had connection with them. Even Now, the constant local spread has led to the early epidemics in all states around the world [1][2]. COVID -19 cases first reported in the Persian Gulf region were linked to people who went from Iran and Iraq to Bahrain and Kuwait.
COVID-19 is presently being fought with all of Iran's might, but control of the sickness has become very difficult concerning the infection's spread across the nation [3]. The human respiratory syncytial virus (RSV) is a pneumovirus belonging to paramyxoviridae group. This negative-sense single-stranded RNA virus is one of the highly widespread viruses to infect children globally and progressively is identified as a critical pathogen in adults, particularly the old subjects. Upper respiratory symptoms are the most common clinical signs in this illness. RSV commonly affects children, causing bronchiolitis, a lower respiratory tract infection with moderate breathing problems, and may rarely progress to pneumonia, respiratory failure, apnea, and decease [4]. Viral detection methods have significantly advanced in current years, as using molecular diagnostic techniques have considerably improved the capability to detect viruses [5].
The association between contamination with several respiratory viruses and the seriousness of the disease has not been well recognized. In studies that examined respiratory diseases by nucleic acid amplification techniques evaluating a significant number of viruses, such frequency was greater than 40% [6][7]. A study in Italy showed that coinfection of RSV and metapneumovirus was shortened the hospitalization and hypoxia, in comparison to RSV infection alone [8]. A French research similarly observed shorter duration of hospitalization in newborns with coexistent RSV and rhinovirus infection contrasting to single RSV infection [9].
On the other hand, respiratory viral diseases have long been established as major causes of death and disability in adults and elders. The precise method by which these pathogenic agents, which ordinarily produce self-limited illnesses in healthy young individuals, are connected to severe aging symptoms is unknown [10]. Blunted immune response accompanied by agingrelated physiologic changes may play a role in this problem. Influenza virus has usually got the greatest consideration followed by RSV and parainfluenza viruses that have been associated with serious illness in old subjects [10]. Determining the prevalence of coinfection of respiratory viruses with COVID-19 is essential to defining its true clinical influence. According to early reports from China, co-infection with other respiratory viruses was uncommon [11]. A research in the United States looked at coinfection in adults with COVID-19 and found that rhinovirus/enterovirus was the most prevalent (6.9%), followed by RSV (5.2%) [12]. Since the presence of more respiratory infections may increase the risk of morbidity among patients infected by COVID-19, well understanding of coinfection is critical. Hence, we report co-infection rates between SARS-CoV-2 and RSV in Shiraz, south of Iran.

Ethical Statement
This cross-sectional study was carried out based on the Whole-mouth saliva, parotid saliva, buccal, and palatal exfoliates were collected and processed for RSV amplification. The volunteers were asked to sit in a comfortable position and were asked to rinse their mouths with bottled water to remove food debris.
A trained nurse and a final-year dental student collected the throat swabs, NPAs, and saliva samples by using standard operating methods from patients who agreed to participate in the study. Demographic and clinical information were collected from each patient using a standardized data form.
The specimens were placed into a vial containing 1 mL of transport media that composed of phosphate buffer saline, glycerol, anti-biotic and anti-fungal. They were transported to the Valfagre specialty laboratory, where they were tested by using real-time PCR for RSV. The samples were divided into aliquots and stored at -80°C until use.

Viral RNA detection
Viral nucleic acid was extracted from 0.2 mL of each respiratory specimen using the high pure viral nucleic acid extraction kit (Roche Diagnostics, Mannheim, Germany). Complementary DNA (cDNA) was synthesized using the first strand cDNA synthesis kit (Thermo Scientific, Waltham, MA USA). RNA integrity was confirmed by amplifying a genomic β-globin sequence as a reference gene.
We developed a highly sensitive and specific PCR using TaqMan technology, virus-specific primers, and probes designed to detect RSV (Table 1) Table 3). The polymerase chain reaction product on gel electrophoresis to detect RSV produced a band of about 379 bp (Figure 1). The chi-square test did not show a significant difference between RSV infection in COVID -19 patients and healthy subjects (p= 0.056).   Zandi et al [16] reported that in cases with sever COVID-19; the higher rate of coinfection especially with RSV could be seen than in mild forms. So prevention of the contamination may influence the prognosis especially in children. These findings differ with us due to our sample size and age [16].
Calvo et al. [15] compared simultaneous coinfection of RSV and rhinovirus in hospitalized children. Elevated fever degrees and hypoxia were more frequent in the RSV-rhinovirus coinfected cases than in specific virus infections. The adult and young adult patients with COVID-19 were compared with healthy subjects [15].
Goka et al. [17] did not find any finding to indicate the symptoms varied obviously between coinfection compared to single viral infections. Because the various characteristics of the virus may produce a bias when analyzing the outcomes, they reported that the results of the surveys vary in various populations. COVID-19 is a newly diagnosed disease with more unexplained findings that need more evaluation [17]. Costa et al. [18] examined a large group of children with rhinovirus diseases and found that more than 50% of them had coinfection, mostly with RSV with more severe symptoms.
All cases had a severe respiratory disorder and the diagnosis of pneumonia was common [18].
Semple et al. [19] discovered a powerful correlation between the coinfection of human metapneumovirus and RSV in newborns with bronchiolitis and dual infection presented a ten-fold increase in relative risk of admittance to a pediatric intensive-care unit. The current research focused on the co-infection of SARS-COV-2 and RSV in older communities. Coinfection of RSV had a seasonality that exactly matches up with the severity of the disease [19]. There are limited researches regarding viral superinfection with COVID-19 in the literature.
Therefore, comparing and analyzing the findings are so complicated and need a long time and more effort.
Kumar et al. [20] presented a patient with a coinfection of tuberculosis and SARS-COV-2 with serious systemic symptoms and an absence of radiological findings detailed to TB. This is a major warning for the Iranian population that TB has increased in many parts of our country [20].
Langford et al. [21] determined the frequency of Kim et al. [12] study demonstrated that among 116 SARS-CoV-2 studied cases, 5.2% were infected with RSV, which is lower than our report [12]. This finding is not in accordance with us, the difference may be associated with the seasonal variation of the virus and the personal characteristic, immune response baseline, and ethnicity of the subjects.
The study of Pinky and Dobrovolny [14] [24][25], with no available difference in clinical symptoms in patients with and without this co pathogen.
One of the powers of our survey is that we evaluated viruses in pairs, but the total of infected cases to assess was not significant enough to permit us to make ideal decisions. To the best of our knowledge, our report has a larger but non-appropriate number of cases in pairs of viruses up to now.
To complete, the potential co-infection of RSV in COVID-19 patients with clinical characteristics and patterns of the corona should always be regarded.
Moreover, assessing more signs and symptoms should be considered.
Our samples were limited to one location (Shiraz).
On the other hand, our country's restrictions also affect the availability of kits, and proper examination tests, lack of proper protective devices, and multiply test types of equipment. On the other hand, no available data was about the prevalence of RSV in Shiraz and data of COVID 19 epidemiology in Iran. However, these results indicate that standard testing for non-SARS-CoV-2 respiratory pathogens in the course of the COVID-19 pandemic is questionable to offer clinical assistance unless a definite cause would alter disease management.

Conclusion
The outcome of present research showed that concurrent